Animal venoms are complex mixtures of toxins with single species containing hundreds of components including neurotoxins with exquisite target specificity. They are of interest for potential discovery of toxins with novel target specificities that could be used for drugs or neurophysiological research. They also are of interest because of the damage to humans caused by venoms of some species. Spiders, because of the complexity of their venoms (200- 1000 toxins in a single spider) and large number of species (~42,000 described!), are predicted to contain the largest pools of toxin diversity of any venomous lineages, yet their venoms remain little explored. Moreover, very few analyses have been done on the evolutionary dynamics of spider venom toxins. Insight into evolutionary mechanisms influencing venoms can serve as a guide for toxin discovery and development of antivenom therapies. The goal of this project is a comparative evolutionary analysis of the full set of venom components ("venomes") among species of sicariid spiders. Sicariids include the notorious brown recluse and their relatives whose bites are capable of causing dermonecrotic lesions and systemic effects in humans. There are ~125 species of sicariids and differences among them represent 100 million years of evolution since their common ancestor. They are also in a spider suborder (Haplogynes) within which very little is known about venoms. In Specific Aim 1 we will use combined transcriptomic and proteomic approaches to characterize the complement of venom-expressed proteins and peptides in representatives of all major lineages of Sicariidae and two outgroups. In Specific Aim 2 we will use computational biology approaches to analyze evolutionary dynamics (duplication patterns, evidence of selection, structural motifs) of individual lineages of toxins and compare them to infer the degree of conservation and variation among toxins in this lineage. These data will provide a comprehensive view of protein and peptide components in venoms of brown recluse and their relatives, facilitating assessment of the possible contribution of currently uncharacterized toxins to the human response to envenomation. Patterns of similarity and differences among species will help with understanding relative risks associated with bites across species, and with developing diagnostics and treatments that are effective for bites from species across the entire genus. The data will also be a platform for discovery of venom toxins with unique neurophysiological activities. Preliminary data indicate we are likely to discover toxins that are significantly divergent from those that are currently known. PUBLIC HEALTH RELEVANCE: The data collected in this work will provide a comprehensive view of protein and peptide components in venoms of brown recluse and their relatives, facilitating assessment of the possible contribution of currently uncharacterized toxins to the human response to envenomation. Patterns of similarity and differences among species will help with understanding relative risks associated with bites across species, and with developing diagnostics and treatments that are effective for bites from species across the entire family. The data will also be a platform for discovery of venom toxins with unique neurophysiological activities